Transposon tagging populations are used in modern plant genomics research to identify genes affecting traits of agronomic or general importance by reverse genetics approaches.
They represent complementary tools for gene discovery, as transposon populations are commonly used to identify the gene responsible for an observed phenotype, the so-called forward genetics approach. This is distinguished in the art from the reverse genetics approach wherein, mutational events are identified in sequences (genes) of interest. The rate-limiting step for the methods is the screening work associated with identification of the individual carrying a mutation in the gene or sequence of interest. Below, the principles of transposon populations and the screening methods are described in more detail and more efficient screening methods are presented which increase the value of these tools for gene-discovery.
Transposons are mobile genetic elements occurring, naturally or engineered, at multiple copies in the genome. They are unstable as their position in the genome can change by excision and insertion at novel sites, usually at any given moment in the life cycle. Transposon populations are valuable for gene-discovery because they can disrupt gene function if they insert in gene sequences or their regulatory regions. The sequences of many transposons used in plant breeding are known, but once a plant with an interesting phenotype is observed, it is not known which gene is affected by transposon insertion. It is, in general, also not known if and if so, which, transposon is responsible for the phenotype. Depending on the organism and transposon, copy numbers of transposons in transposon populations range from several tens to hundreds of transposon per plant.
Current screening methods for analysis of transposon-induced phenotypic mutant sequences include linked-PCR based methods in order to obtain flanking sequences from sequence-specific transposon integration sites. A limitation of linker-PCR is that determination of flanking sequences requires band-excision from sequencing gels, which is time-consuming, difficult to automate and relatively low-throughput (not easily adaptable to thousands of bands).
Screening transposon populations would be improved if a simple method would be available to collect flanking sequences of all or at least part of the transposons, integrated in the genome. Here we seek to provide an efficient approach to analyse and use insertion events in preferred sequences.